CN105007933A - Selective NOx-1 inhibitor peptides and uses thereof - Google Patents

Selective NOx-1 inhibitor peptides and uses thereof Download PDF

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Publication number
CN105007933A
CN105007933A CN201480003978.1A CN201480003978A CN105007933A CN 105007933 A CN105007933 A CN 105007933A CN 201480003978 A CN201480003978 A CN 201480003978A CN 105007933 A CN105007933 A CN 105007933A
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peptide
seq
skin
compositions
disease
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H·R·雷兹瓦尼
F·梅祖里哀
A·泰伊伯
L·G·阿尔富什
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Bordeaux, University of
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Bordeaux
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Institut National de la Sante et de la Recherche Medicale INSERM
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Abstract

The present invention relates to novel peptides, compositions and methods for the prevention and/or treatment of pathological conditions and diseases associated with NADPH oxidase 1 (Nox1) activity, and/or increased reactive oxygen species (ROS) production. The novel peptides are thus particularly useful for treating and/or preventing cancer, atherosclerosis, angiogenesis, and aging.

Description

Selective N ox1 inhibitor peptide and uses thereof
Technical field
The present invention relates to and produce to nadph oxidase 1 (Nox1) activity and/or reactive oxygen species (reactive oxygen species) (ROS) new type of peptides, compositions and the method that increase relevant pathological conditions and disease for preventing and/or treating.Therefore, described new type of peptides is specially adapted to treat and/or prevent cancer, atherosclerosis, blood vessel generation and aging.
Background technology
Nox1 generates ROS in physiological conditions with in some pathological conditions.Known ROS causes damage in vivo, is therefore correlated with from many different diseases.Also known nadph oxidase is the source of oxidative stress, which results in the cause of disease of many diseases.The expression of known Nox1 is correlated with from many different diseases (comprising cancer).
Virose therapeutic choice is not had to be obviously important for therapeutic agent.Specifically, in treatment of cancer, preferentially kill cancer cell and do not have toxicity to be one of most important Consideration to normal cell.
Before making the present invention, identify the inhibitor of some nadph oxidases (Nox), but not yet develop specific Nox inhibitor up to now.
Also tested some can the inhibitor peptides of penetration cell, such as gp91ds-tat peptide, PR-39, and Rac inhibitor peptides.Gp91ds-tat peptide is designed to carry out specificity by simulation Nox2 sequence and suppresses Nox2, and it is important that described Nox2 sequence is considered to the interaction with p47phox.But because the region of described peptide targeting is homology in other Nox isoforms, therefore, described peptide lacks specificity.In addition, described peptide is less potent inhibitors, and it suppresses the ROS of neutrophil cell generation to reach 25% when 50mM.
Recently, GenKyoTex company uses pyrazolo-pyrido-diazepine ,-pyrazine and-oxazine derovatives to develop more specific Nox inhibitor, its concrete targeting Nox1 and Nox4 enzyme.GenKyoTex is using micromolecule GKT137831 to carry out I clinical trial phase at present, is used for the treatment of diabetic nephropathy.Also carry out the model that preclinical test is used for the treatment of diabetic nephropathy, atherosclerosis, idiopathic pulmonary fibrosis, hepatic fibrosis and blood vessel occur.But, although GKT137831 be specificity for Nox1/Nox4, it also shows affinity to Nox2, Nox3 and Nox5, therefore has low selectivity.
Other compounds are used for many years, comprise 4-hydroxy-3-methoxyacetophenone (apocynin), diphenylene iodine (DPI), 4-(2-amino-ethyl)-benzene sulfonyl fluorine hydrochlorate (AEBSF) and 1-(2-amino-4-hydroxy-6-pteridinyl)-1,2,3-propanetriol.
4-hydroxy-3-methoxyacetophenone extracts from Rhizoma Picrorhizae (Picrorhiza kurroa), and it can stop the formation of active oxidation combined enzyme agent.But the inhibitory action of 4-hydroxy-3-methoxyacetophenone is controversial.In fact, proved 4-hydroxy-3-methoxyacetophenone and non-specific for nadph oxidase, but affected other events, such as thromboxane A2 is formed and the induction of AP-1 transcription factor, and 4-hydroxy-3-methoxyacetophenone is antioxidant but not Nox inhibitor.
Diphenylene iodine (DPI) is the nonspecific inhibitor of all flavo-enzymes, therefore, it is possible to suppress Nox enzyme, but also can suppress xanthine oxidase and cytochrome P 450 enzymes.Except being nonselective, also prove that DPI has toxicity.It or acetylcholinesterase and butyrylcholine esterase and inner Ca 2+the inhibitor of pump, except the toxicity that it is intrinsic, this also result in the major concern to its application.
Prove that amino-ethyl-benzene sulfonyl fluorine (AEBSF) stops flavin cytochrome b 558with p47 phoxin conjunction with O-in the activation of, nadph oxidase and macrophage 2what produce excites.But AEBSF is serpin mainly; Therefore it with more high efficiency protease inhibition (such as Chymotrypsin, kallikrein, fibrinolysin, thrombin and trypsin), and can have other effects many.
---it is by the indirect Nox inhibitor of misleading ground called after---the upstream signal transduction path of interference effect Nox enzyme that proved some other non-specific molecules.The inhibitor (such as benzo (b) pyrans-4-ketone derivatives, the S17834 of Shionogi Pharma) of the sulfo-triazolopyrimidine compound VAS2870 that the PDGF-dependency Src that these molecules comprise suppression Nox enzyme activates, angiotensin-convertion enzyme inhibitor (ACE inhibitor), Ang II receptor blocking agent (ARB), phosphodiesterase, eicosanoid, corticosteroid, map kinase, Protein kinase C.
Also has more and more report to using for the siRNA method of Nox enzyme.Specifically, developed some RNAi to express to reduce Nox1.But, use RNAi to be still in the junior stage as the Results of the mankind, and time several years may be needed to optimize.Unfortunately, the specificity of a small amount of siRNA to Nox isoform is only really tested.Without any the gratifying siRNA method for Selective depression Nox, particularly Nox1.
Therefore, the compound developed up to now directly cannot play a role and block described enzyme, but disturbs upstream Signal Transduction Pathways or work as antioxidant or ROS scavenger.But unique compounds that directly can block described enzyme lacks selectivity and suppresses other enzymatic activitys.They also have low effect and bioavailability, thus cannot pharmacologically prove them in vivo using Nox as therapeutic targets.Some micromolecule of Nox enzyme and inhibitor peptides are used in experimentation, but the problem of specificity and toxicity limits the candidate of any existing compound as drug development.
Consider a large amount of disease targets recorded in nearest research, find inhibitor useful in the novel clinical of Nox enzyme to be vital.But maximum challenge is the inhibitor peptides finding to act on specifically single Nox isoform.
Applicant has accepted and has successfully solved this challenge.In fact, the invention provides directly the new type of peptides inhibitor that also specific inhibition nadph oxidase 1 (Nox1) assembles, it does not suppress other nadph oxidases, particularly Nox2, Nox4, Nox5, Duox1 and Duox2.Nox1 relates to generation and the development of spectrum of diseases, and described disease comprises cancer, aging, neurodegenerative disease and cardiovascular disease, and therefore Nox1 represents important therapeutic targets.Therefore, new type of peptides of the present invention is also particularly advantageous, removes or antioxidant activity and they do not suppress other ROS to originate because it does not have non-specific ROS.Compared with RNAi technology, described inhibitor peptides very specifically for its target protein, thus reduces the probability of effect of missing the target.Compared with chemical inhibitor, peptide of the present invention does not show toxic action in vivo.In addition, peptide of the present invention is easy to design and produces.
Summary of the invention
Therefore, the invention provides the peptide itself comprising aminoacid sequence, described aminoacid sequence comprises at least 7 to 35 continuous amino acids, comprises a part for following aminoacid universal sequence: X1-P-X2-X3-P-X4-R (SEQ ID NO:1), and wherein X1 is A, P or Q; X2 is P, T, V, A, S, C, M or K; X3 is L, I, V or A; X4 is T, V, S, A, M.Described peptide is specially adapted to prevent and/or treat and/or reactive oxygen species (ROS) active to Nox1 and produces and increase relevant pathological conditions or the method for disease.
The present invention relates to the new selective peptide that Selective depression Nox1 activates, be referred to as Nox1 inhibitor peptide hereinafter.Therefore these new type of peptides are suitable as medicine, as light protection reagent and/or as defying age reagent.They are optionally conjugated to cell-penetrating peptides or increase the reagent (cell carrier) that described peptide accumulates in cell.
According to the first embodiment, the present invention relates to the compositions of the Nox1 inhibitor peptide of the present invention including effective amount, and comprise the treatment Nox1 inhibitor peptide of effective dose and the pharmaceutical composition of pharmaceutically acceptable excipient (vehicle or excipient), and cosmetic composition.
In the method that novel Nox1 inhibitor peptide of the present invention and compositions can be advantageously used in the disease treating and/or preventing Mediated by Free Radicals---being more specifically nadph oxidase 1/Nox1 relevant disease---, described disease comprises cardiovascular disease, angiogenesis dependent disease, respiratory system disease, dermatosis, neurodegenerative disease, allergy and autoimmune disease, gastrointestinal disease, cancer and the disease relevant to impaired metabolism.The most especially, they be applicable to treat and/or prevent blood vessel and cardiovascular disease, Cancerous disease (such as colon cancer and skin carcinoma) and dermatosis, UV skin injury, the skin disorder caused because of aging, ROS induction skin injury, cross presenility and/or ultraviolet (UV A or UV B) expose after the method that generates of cutaneous tumor in.
According to the second embodiment, the present invention relates to the method for the generation of level and/or the inhibit activities oxygen clusters (ROS) reducing reactive oxygen species (ROS) in experimenter in need, particularly selectivity reduces and/or suppresses the method for nadph oxidase 1 (Nox1) activity, and it comprises the novel Nox1 inhibitor peptide or compositions that give to treat effective dose.
According to the 3rd embodiment, the present invention relates to the disease the treating and/or preventing Mediated by Free Radicals method of---being more specifically nadph oxidase 1/Nox1 relevant disease---, described disease comprises cardiovascular disease, angiogenesis dependent disease, respiratory system disease, dermatosis, neurodegenerative disease, allergy and autoimmune disease, gastrointestinal disease, cancer (such as colon cancer and skin carcinoma) and the disease relevant to impaired metabolism.Method of the present invention is also applicable to the cutaneous tumor after treating and/or preventing dermatosis, UV skin injury, the skin disorder caused by aging, the skin injury of ROS induction, excessively presenility and/or ultraviolet (UV A or UV B) exposure and generates.
According to the 4th embodiment, the present invention relates to the method for the peptide prepared described new type of peptides and be optionally conjugated to cell-penetrating peptides or cell carrier.
Accompanying drawing explanation
Figure 1A and 1B: show the cytotoxic effect using trypan blue exclusion mensuration and MTT to be determined at often kind of peptide that normal primary Human keratinocytes is tested.As shown in Figure 1A and 1B, adopt peptide A (SEQ ID NO:41), the peptide B (SEQ ID NO:42) of variable concentrations and PEPC (SEQ ID NO:43) handler's keratinocyte, and use Trypan Blue dye (A) and MTT mensuration (B) to carry out the vitality (viability) of after measurement processing 24,48 and 72 hours.Relative to untreated cell (NTC), the vitality percent of treated keratinocyte is normalized.
Fig. 2 A and 2B: show and use CM-H2DCF-DA probe, in the primary Human keratinocytes of employing 10 or 50 μ Μ inhibitor peptides process and the relative ROS level of measurement in HT29 (human colon adenocarcinoma cell line).Adopt 10 μ Μ peptide A (SEQ ID NO:41), peptide B (SEQ ID NO:42) and PEPC (SEQ ID NO:43) to process cell, and within 24 hours, measure ROS level after treatment.Relative to untreated cell (NTC), ROS level is normalized (* P<0.05).According to Fig. 2 A, in the cell adopting peptide A (SEQ IDNO:41) and peptide B (SEQ ID NO:42) to process, observe ROS level significantly reduce.According to Fig. 2 B, in HT29 cell, the measurement of ROS level shows, adopts the process of peptide A and B to cause ROS level significantly to reduce, shows the specific inhibitory effect of these peptides to Nox1 activity.
Fig. 3 A, 3B and 3C: show and use the shRNA for the lentivirus mediated of Nox1 and Nox2 to express suppression to endogenous Nox1 and Nox2 protein expression.Adopt different shRNA (shNox1, shNox2 and contrast shCtrl) keratinocytes of transduceing.Transduction efficiency is checked by western blot analysis.Fig. 3 A shows shNox1 and shNox2 and stably suppresses Nox1 and Nox2 of more than 80% to express respectively.Transduction efficiency is checked by western blot analysis.In employing Nox1 inhibitor peptide A and latter 24 hours of control peptide C process, measure relative ROS level (3B) and Nox activity (3C).Relative to the cell of the PEPC process of correspondence, ROS level is normalized.Nadph oxidase activity is measured by the relative light units (RLU) of every micrograms of protein.*P<0.05。According to Fig. 3 B, the measurement of ROS level shows, in the cell of shNox1 transduction, adopt the steady-state level of process to ROS of inhibitor peptide A not act on, show that peptide A generates with the dependent ROS of the efficiency of very high (close to 100%) and specific inhibition Nox1.In fig. 3 c, in the cell used or do not use shCtrl and shNox1 of Nox1 inhibitor peptide A process to transduce, nadph oxidase activity is measured.
Fig. 4 A, 4B and 4C: show Nox1 inhibitor peptide A and ROS is produced and the suppression of nadph oxidase activity, and therefore to the photoprotection of people and Mus keratinocyte.Adopt the keratinocyte that 10 μ Μ Nox1 inhibitor peptide A or contrast scrambled peptide (scramble peptide) C process are separated from people's biopsy samples (Ker people) with mouse skin (Ker mice).Within 24 hours, measure ROS level (4A) and nadph oxidase activity (4B) after treatment.The nadph oxidase activity adopted in the people of contrast scrambled peptide C process and mice keratinocyte is forced to be set as 100%.Then assessment adopts the suppression percent (4C) of the rear nadph oxidase activity of Nox1 inhibitor peptide A process.
Fig. 5 A and 5B: to show in mouse skin peptide A to the effect of Nox1 activity.Adopt following various dose Local treatment SKH-1 mice: 0.4,3 and 12mg/kg (every dosage 3 mices) Nox1 inhibitor peptide A.2h, 24h, 48h and 72h gather Skin biopsy sample after treatment, and measure nadph oxidase activity (RLU/ μ g protein).Relative to untreated (NT) mice result be normalized and be expressed as the suppression percent (%) of Nox activity.(B) 3 and 12mg/kg peptide A (every dosage 2 mices) Local treatment mice is adopted.10min after process, makes mice be exposed to UVB (150mJ/cm 2).Skin biopsy sample is gathered at 2h, 24h.46h after first process, adopts the peptide A of same concentrations again to process mice and 10min makes mice be exposed to UVB after treatment.Then Skin biopsy sample is gathered at 2h, 24h (after being namely respectively first process 48h and 72h).Measure nadph oxidase activity (RLU/ μ g protein), and be normalized relative to untreated (NT) and non-irradiated mice.
Fig. 6 A, 6B and 6C: show the photoprotection that the squamous cell carcinoma (SCC) of peptide A to the UVB induction using SKH-1 hairless mouse to carry out is induced.Adopt 3mg/kg peptide A or control peptide Local treatment mice and after 10min, irradiate mice with UVB, 3 times weekly.In 29 weeks, (6C) assesses quantity (6A) and the size (6B) of tumor once in a week.Gross tumor volume distribution is assessed the 29th week time.Obviously, the quantity and the size that give the tumor of UVB induction in the SKH-1 mice of peptide A in local significantly reduce.
Fig. 7: show in the SKH-1 mice adopting peptide A and control peptide process, in the tumor of non-lotus tumor skin and UVB induction, nadph oxidase activity all significantly reduces.After treatment 29 weeks time put to death mice.What process from adopting peptide A or excipient (vehicle) gathers non-lotus tumor skin and tumor through irradiating mice.Measure the nadph oxidase activity (being expressed as RLU/ μ g protein) in the tumor of non-lotus tumor skin and UVB induction.Force the Nox activity taken from the non-lotus tumor skin of the mice that excipient (vehicle) processes and tumor to be set as 100%, then assess in the relative suppression of non-lotus tumor skin with Nox activity in tumor.Then, result is expressed as the average percentage of the mice processed through excipient (vehicle).
Fig. 8 A with 8B: show the peptide A without tat and can equally with peptide A effectively suppress Nox active.In addition, the less peptide derived from peptide A suppresses nadph oxidase active effectively.Adopt following the indicated peptide process keratinocyte of 10 μ Μ: out of order control peptide P1 (SEQ ID NO:57), there is the peptide A (SEQ ID NO:41) of tat sequence or not there is peptide A (SEQ ID NO:20), comparatively small peptide P7 (SEQ ID NO:2) and the P8 (SEQ ID NO:56) (Fig. 8 A) of tat sequence.24h measures nadph oxidase activity (RLU/ μ g protein) after treatment, and is normalized (Fig. 8 B) it relative to the cell of out of order process.
Fig. 9 A and 9B: show at sound (the proficient) (XPC of young and old XPC + /+) and XPC defect (XPC -/-) mice Skin biopsy sample in presenilin (progerin) express.Young mice was 4 monthly ages, and aged mice is 1.5 years old age.(A) presenilin in the total protein extract of Skin biopsy sample is assessed by western blot analysis.Use beta-actin as loading control (loading control).(B) beta-actin is adopted to be carried out quantitatively and normalized by the protein band corresponding to presenilin.The relative value relative to young wild-type mice Midst density is expressed as by from the average density ± SD of 6 mices in every group.
Figure 10 A, 10B and 10C: show the effect that XPC defect is expressed Nox activity and OXPHOS complex.(A) young and old XPC is measured + /+and XPC -/-nox activity (RLU/ μ g protein) in the Skin biopsy sample of mice.Relative to young wild-type mice, result is normalized.(B) young and old XPC perfect with deficient mice in be assessed the expression of OXPHOS complex by western blot analysis.(C) beta-actin is adopted to carry out quantitatively and normalized the band corresponding to different OXPHOS complex.The relative value relative to young wild-type mice Midst density is expressed as by from the average density ± SD of 6 mices in every group.
Figure 11 A and 11B: show Nox1 inhibitor peptide A (InhNOX) to young XPC + /+and XPC -/-the effect that in the Skin biopsy sample of mice, presenilin is expressed.Adopt 3mg/kg Nox1 inhibitor peptide A (InhNOX) or control peptide (excipient (vehicle)) Local treatment 1 monthly age mice (often organizing 6 mices), process 3 times weekly, continue 3 months.(A) presenilin in the total protein extract of Skin biopsy sample is assessed by western blot analysis.Use beta-actin as loading control.(B) beta-actin is adopted to carry out quantitatively and normalized by the protein band corresponding to presenilin.The relative value relative to young wild-type mice Midst density is expressed as by from the average density ± SD of 6 mices in every group.
Figure 12 A and 12B: show Nox1 inhibitor peptide A (InhNOX) to young XPC + /+and XPC -/-the effect that in the Skin biopsy sample of mice, OXPHOS complex proteins is expressed.Adopt 3mg/kg Nox1 inhibitor peptide A (InhNOX) or control peptide (excipient (vehicle)) Local treatment 1 monthly age mice (often organizing 6 mices), process 3 times weekly, continue 3 months.(A) expression of OXPHOS complex is assessed by western blot analysis.Use beta-actin as loading control.(B) beta-actin is adopted to be carried out quantitatively and normalized by the band corresponding to different OXPHOS complex.The relative value relative to young wild-type mice Midst density is expressed as by from the average density ± SD of 6 mices in every group.
Detailed description of the invention
The present invention relates to new selective peptide, it can be assembled by specific inhibition nadph oxidase 1 and therefore block nadph oxidase 1 and activates and optionally suppress Nox1 to activate, and it protects the application of reagent as medicine and light.Therefore the present invention relates to treatment and beauty treatment two kinds of methods.Hereinafter described peptide is called Nox1 inhibitor enzyme.
In order to using the protein in cytosol as target, extra modification can make peptide penetrate into cell.The example of successful methods adds to be rich in arginic sequence or tat sequence.Assembling with Nox1 the potential ability competed by interacting with Nox1 and have selected peptide of the present invention based on them.
Individuality to be treated comprises humans and animals, preferred mammal, such as rodent and primate.Described individuality can be non-human animal or non-human mammal.Described individuality can suffer from herein mentioned by any disease, or be in develop described disease risk in.For human individual, it is at least 50 years old age, such as, be at least 60 or 70 years old age.
Peptide of the present invention is from SEQ ID NO:44 (the PRR region of NoxO1) or SEQ IDNO:45 (the SH3 region of NoxA1).They generally include 5-35 the aminoacid from SEQ ID NO:44 or SEQ ID NO:45, or 5-20 aminoacid, or 7-20 continuous amino acid.SEQ ID NO:44 is PPTVPTRPSPGAIQSRCCTVTRRAL, SEQ ID NO:45 is QVVAQHSYSAQGPEDLGFRQGDTVDVLCEEPDVPLAVDQAWLEGHCDGRIGIFPKC FVVPAGPRM.
Peptide of the present invention generally includes two different sequences.One in these is commonly called " part " in this article, and it is responsible for the therapeutic activity of peptide described herein.Described part normal length is 5-20 aminoacid, or 7-20 aminoacid, such as 7-19,7-18,7-17,7-16,7-15,7-14,7-13,7-12,7-11,7-10; Preferred 7-18,7-15,7-10; About 7 aminoacid; Or 8-18,8-15,8-10 aminoacid.
Second in described peptide different sequence, or the remainder of described sequence, can be the characteristic that described peptide provides other favourable.This Part II of described peptide can not exist, and therefore in one embodiment, described peptide only gives " part " sequence composition for the treatment of characteristic by mentioned earlier, and namely the length of described " part " is identical with the length of described peptide.The normal length of described second sequence is 0-31 aminoacid, and such as length is 5-30,7-30,10-25 or 15-20 aminoacid.Described second sequence can be cell-penetrating peptides that is identical with any concrete cell-penetrating peptides mentioned by this paper or homology.Obviously, extra " second " sequence can all be there is at the N-terminal of described " part " and C side.
The normal length of described peptide is 7-35 aminoacid, or 7-30 or 7-20 or 7-15 or 7-10, or about 7,8,9 or 10 aminoacid, and preferred length is 7,8 or 9 aminoacid, and most preferably length is 7 aminoacid.
Described peptide or part can be maybe to comprise the sequence represented with following aminoacid general formula:
X1-P-X2-X3-P-X4-R(SEQ ID NO:1)，
Wherein X1 is A, P or Q;
X2 is P, T, V, A, S, C, M or K;
X3 is L, I, V or A; And
X4 is T, V, S, A or M.
Described peptide or part can be maybe to comprise any sequence listed in following table 1, can be maybe its congeners:
Table 1
PPTVPTR(SEQ ID NO：2)
PPSVPTR(SEQ ID NO：3)
PPMVPTR(SEQ ID NO：4)
PPCVPTR(SEQ ID NO：5)
PPPVPTR(SEQ ID NO：6)
PPAVPTR(SEQ ID NO：7)
PPVVPTR(SEQ ID NO：8)
PPTVPSR(SEQ ID NO：9)
PPTVPMR(SEQ ID NO：10)
PPTVPVR(SEQ ID NO：11)
PPTVPAR(SEQ ID NO：12)
QPTAPTR(SEQ ID NO：13)
PPTLPTR(SEQ ID NO：14)
PPTIPTR(SEQ ID NO：15)
PPTAPTR(SEQ ID NO：16)
PPKVPTR(SEQ ID NO：17)
QPTVPTR(SEQ ID NO：18)
APTVPTR(SEQ ID NO：19)
According to another embodiment, described peptide or part can be maybe to comprise any sequence listed in following table 2 and table 3, can be maybe its congeners:
Table 2
PPTVPTRPS(SEQ ID NO：20)
IQSRCCTVT(SEQ ID NO：21)
PTVPTRP(SEQ ID NO：22)
TVPTRPS(SEQ ID NO：23)
RPSPGAI(SEQ ID NO：24)
SPGAIQS(SEQ ID NO：25)
PGAIQSR(SEQ ID NO：26)
GAIQSRC(SEQ ID NO：27)
AIQSRCC(SEQ ID NO：28)
IQSRCCT(SEQ ID NO：29)
QSRCCTV(SEQ ID NO：30)
SRCCTVT(SEQ ID NO：31)
Table 3
QVVAQHSYSA(SEQ ID NO:32)
QGPEDLGFRQ(SEQ ID NO:33)
LGFRQGDTVD(SEQ ID NO:34)
GDTVDVLCEE(SEQ ID NO:35)
VLCEEPDVPL(SEQ ID NO:36)
PDVPLAVDQA(SEQ ID NO:37)
AVDQAWLEGH(SEQ ID NO:38)
According to another embodiment, present invention also offers peptide or the part of the aminoacid sequence comprising at least 7-35 continuous amino acid, a 7-30 continuous amino acid or 7-20 continuous amino acid, described aminoacid sequence can be identical with the fragment of SEQ ID NO:44 (the PRR region of NoxO1) or SEQ ID NO:45 (the SH3 region of NoxA1) or have homology.Be applicable to homology level by following about the part of homology in discuss, and be applicable to homology level comprise such as its analog or examples of conservative variations.The sequence for the treatment of part or peptide preferably has the homogeneity of 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:44 or SEQ ID NO:45.Described peptide optionally produces increase relevant pathological conditions or the method for disease for preventing and/or treating and/or reactive oxygen species (ROS) active to Nox1.The length that the length of described peptide is preferably 7-15 aminoacid and/or described part is preferably 7-10 aminoacid, and more preferably length is about 7 aminoacid.Most preferably, described peptide is identical with at least 7 continuous amino acids of SEQ ID NO:44 (the PRR region of NoxO1) or SEQ ID NO:45 (the SH3 region of NoxA1), or has the homogeneity of at least 80% or 85% or 90% or 95% with SEQ ID NO:44 or SEQ ID NO:45.
Homologous sequence
Be referred to homologous sequence herein.Described homologous sequence can represent treatment " part " sequence in described peptide or second (residue) sequence in peptide of the present invention.Described homologous sequence can with any concrete sequence homology mentioned by this paper.Compared with original series, congener has one or more (such as at least 2,3,4 or 5) to be added and/or lacks and/or displacement.Described homologous sequence can comprise the amino acid identities of at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 95%, 96%, 97%, 98% or at least 99% in the whole length of original series or on 4,6,10,15 or 20 aminoacid of original series.
Term " sequence iden " typically refers to and uses ClustalW (Thompson et al., 1994, above-mentioned) and following parameter is assessed time there is the sequence of institute's indicating value: pairing alignment parameters (Pairwise alignment parameters)---method: accurately, matrix: PAM, Gap Opening Penalty (Gap open penalty): 10.00, gap extension penalty (Gapextension penalty): 0.10; Many alignment parameters (Multiple alignmentparameters)---matrix: PAM, Gap Opening Penalty: 10.00, postpones homogeneity percent: 30, end gap point penalty: open, room separation distance: 0; Negative matrix: nothing, gap extension penalty: 0.20, residue specificity gap penalty: open, hydrophilic gap penalty: open, hydrophilic residue: G, P, S, N, D, Q, E, K.Be intended to comprise by the identical residue simply derived at the sequence iden of specific resi-dues.
Compared with original series, homologous sequence can have 1,2,3,4,5 or more or the most nearly 10 amino acid replacements.Described displacement is preferably preservative replacement.These can carry out according to following table 4.Aminoacid in the 2nd row phase apposition, preferably the 3rd row mutually colleague in aminoacid can phase double replacement.
Table 4
By any applicable method as known in the art, such as chemosynthesis and/or recombinant DNA technology, prepare peptide of the present invention.Such as, Solid phase peptide synthesis technology (such as, Fmoc) can be used to synthesize peptide of the present invention.In addition, recombinant DNA technology (such as, using antibacterial or eukaryotic expression system) can be used to synthesize described peptide.Then described nucleotide sequence is changed to realize the introducing of relevant amino acid residue (namely by the nucleotide sequence of isolated or synthesized coding described parent peptide (parent peptide), insert or displacement) or remove (namely, disappearance or displacement), the nucleotide sequence of polypeptide of the present invention of encoding can be built.The method of the synthesis of solid-state protein matter and recombinant protein synthesis is as known in the art.Such as, MolecularCloning, A Laboratory Manual (Sambrook et al., 3d Edition, ColdSpring Harmor Press) be known list of references, many applicable technology prepared by the restructuring wherein detailing polypeptide.Can to be natural can be maybe that not known be naturally occurring variant for the variant of peptide.Prepare the non-native variant of peptide by direct synthesis, or such as according to conventional methods by saturation mutagenesis or direct mutagenesis peptide of the present invention all or part of in introduce sudden change at random.Do not rely on preparation method, the ability of suppression Nox1 and/or ROS of obtained variant can be screened to identify variant of the present invention.
Separable and/or purification (or be substantially separated and/or substantially purification) described new type of peptides.Therefore, the invention provides the peptide of the first aspect present invention of the form (such as, being substantially separated from other peptides or impurity) being separated or being substantially separated.Such as, described peptide can be isolated from other peptides produced because of solid phase protein synthetic method.In addition, described peptide can substantially be isolated in other protein always after the cytolysis of recombinant production.The standard method of protein purification (such as, HPLC) can be used with purification peptide of the present invention substantially.Therefore, the goods of peptide of the present invention were preferably at least 90% (by weight) not containing other peptides and/or pollutant, did not more preferably contain other peptides and/or pollutant (such as not containing other peptides and/or pollutant at least about 97% or 98% (by weight)) at least about 95% (by weight).
Term " peptide " amino acid residue not only comprised wherein is the molecule combined by peptide bond (-CO-NH-), also comprises the molecule that wherein peptide bond is inverted.Method as known in the art can be used to prepare described converse to peptide mimics (retro-inversopeptidomimetic).This method comprises the pseudo-peptide that preparation comprises change, and described change relates to the orientation of main chain and non-branched.Comprise NH-CO key but not CO-NH peptide bond converse much better than to the resistance of proteolysis to peptide (Retro-inverse peptide).Equally, can completely without peptide bond, condition uses suitable blank area, and described suitable blank area retains the spacing between the carbon atom of amino acid residue; Particularly preferably there is with peptide bond the blank area of substantially the same CHARGE DISTRIBUTION and substantially the same flatness.Should also be understood that and can block described peptide expediently to help to reduce the susceptibility to outside proteolytic digestion (exoproteolytic digestion) at its N-terminal or C-terminal.Such as, by the N-terminal of described peptide being protected amino, by reacting with amine the C end carboxyl can protecting described peptide with carboxylic acid reaction.Other examples modified comprise glycosylation and phosphorylation.Another kind of possible modification is, can adopt the hydrogen (-NH on methylene group displacement R or K chain amine 2-"-NH (Me) or-N (Me) 2).
Described peptide is modified, such as, to increase or to reduce the Half-life in vivo of described peptide by other modes.Therefore class peptide analogues, D-amino acid derivativges and peptide-class peptide crossbred can be used.Other embodiments of variant polypeptide used in the present invention comprise the D-amino acid form of polypeptide.Use D-aminoacid but not the amino acid whose polypeptide product of L-considerably reduces the undesirable degraded of normal metabolic processes to this kind of reagent, decrease the amount of reagent and the administration frequency of reagent that need to give.
Peptide of the present invention can reduce Nox1 activity, such as, reduce at least 20%, or at least 30%, or at least 35%, 40%, 45%, 50%, 54%, 55%, 56%, 57%, 58%, 59% or 60%.Preferably, described peptide optionally reduces and/or suppresses Nox1 active, but does not suppress the people's nadph oxidase being selected from Nox2, Nox4, Nox5, Duox1 and Duox2.More preferably, peptide of the present invention stops the interaction between NoxO1 and NoxA1 but not the interaction between p47phox and p67phox by specificity, and optionally reduces and/or suppress Nox1 active.Therefore, peptide of the present invention is as described above Nox1 specificity peptide for inhibiting.
The invention still further relates to the method and composition of the damage of the ROS mediation alleviated the skin of experimenter or the nadph oxidase of mucosa or NOX induction.One embodiment of the invention are methods of the damage of the ROS mediation suppressing and alleviate the skin of experimenter or the nadph oxidase of mucosa or NOX induction, it comprises by the delivery of peptides of the present invention of the effective dose in pharmaceutically acceptable carrier to experimenter, and described experimenter suffers the damage to its skin or the nadph oxidase of mucosa or the ROS mediation of NOX induction.The present invention also provides the method for the free radical of the ROS mediation regulating nadph oxidase or NOX induction, and described free radical causes the oxidation tissue damage relevant to senilism.The present invention usually also comprises and being joined in suitable compositions as nutriment and/or pharmaceutical agent by peptide of the present invention.Term " adjustment (modulate) ", " regulating (modulation) " or " regulating (modulating) " comprise rising or the reduction of the activity of any composition (particularly Nox1) of nadph oxidase or NOX system.Be height reactive molecule due to ROS and can produce multiple damage in cell, therefore, ROS vicious cycle is considered to the exponential increase causing oxidative damage in aging course, and this causes the functional decline gradually as aging course feature.
In preferred embodiments, described peptide and compositions thereof protect reagent very effective as light.Sunlight plays an important role to skin, causes too early skin aging, skin carcinoma and many skin changes.Be exposed to the too early skin ageing symptoms that ultraviolet (particularly UVA or UVB) causes 90%.Skin carcinoma is the serious consequence that long term ultraviolet exposes.Ultraviolet radiation from the sun is the Etiological of skin carcinoma.Ultraviolet injury DNA, the i.e. hereditary material of constitutivegene.The growth of Gene Handling Skin Cell and general health.If genetic damage is serious, normal Skin Cell may start to grow with not controlled, disorderly mode, becomes carcinous.Ultraviolet also causes sunburn and makes skin seem presenility and other damages wrinkly.According to the skin cell types that skin carcinoma occurs, there is dissimilar skin carcinoma.Skin carcinoma can be basal cell carcinoma, squamous cell carcinoma or malignant melanoma.
According to the present invention, describe the compositions based on peptide of the present invention, its enhancing is protected the light of UVA and UVB two kinds of radiation.Described compositions can be used for treating the experimenter being easy to the risk being in the relevant skin injury of acute or chronic UV exposure.Described compositions gives by local, oral or intradermal routes.Described compositions can easily be included in the excipient (vehicle) of local application, as the method for skin area active agent being directly administered to needs, described excipient (vehicle) is solution, suspension, Emulsion, oil, emulsifiable paste, ointment, powder, liniment, ointment (salve) etc. such as.In addition, described compositions also can be included in peroral dosage form, such as capsule, tablet, syrup, taffy, chocolate, functional drinks etc.
In preferred embodiments, the invention provides the method relating to the cutaneous tumor after treating and/or preventing long-term UV exposure (UVA or UVB) and occur.In other embodiments; the present invention relates to process skin with the method providing protection to avoid UV radiation; it comprises: will comprise the treatment Nox1 inhibitor peptide mentioned above of effective dose or the compositions of its analog or its examples of conservative variations is administered to skin, wherein said peptide is optionally conjugated to or is connected to increases the reagent that accumulates in cell of described peptide.Most preferably, the peptide had according to the sequence of the aminoacid general formula of SEQ ID NO:1 or any aminoacid sequence as shown in SEQ ID NOs:2 to 42 (preferred SEQ ID NOs:2 to 19) can be used.
In other preferred embodiments, the present invention relates to treatment and/or the care method of skin, mucosa, scalp and/or hair, it comprises the above-mentioned Nox1 inhibitor peptide or its analog or examples of conservative variations that give beauty treatment or medicine effective quantity, and wherein said peptide is optionally conjugated to or is connected to the reagent increasing described peptide and accumulate in cell.Described peptide preferably has the sequence of SEQID NO:1 to 42.In preferred embodiments, the present invention relates to treatment and/nurse because ROS generates the method for the disease of skin, mucosa, scalp and/or the hair caused, disease and/or condition of illness.More specifically, the treatment provided by method and composition of the present invention and/or nursing are light protections, to the protection of the cell DNA of skin, mucosa, scalp and/or hair and/or the reparation of cell DNA.
According to other embodiments, carry out adopting the treatment of compositions of the present invention and/or skin nursing to alleviate, to postpone and/or the sign of anti-aging, photoaging etc. in advance.Particularly, medicable disease states comprises the skin disorder because old and feeble and mistake presenility cause, and wherein peptide of the present invention gives as defying age reagent or bright protective agent.
The present invention also provides the compositions strengthening and protect the light of the skin injury that both UVA and UVB induce, and described compositions comprises the above-mentioned Nox1 inhibitor peptide or its analog or examples of conservative variations for the treatment of effective dose.Described peptide is optionally conjugated to or is connected to the reagent increasing described peptide and accumulate in cell.In preferred embodiments, described compositions provides safety, long-term therapeutic scheme for needing the experimenter of the protection of the light to UVA and UVB two kinds of rays strengthened.Particularly, described compositions can be given the experimenter being easy to because long-term UVA and UVB exposes be in risk of skin cancer.
The present invention relates to the method that selectivity in experimenter in need reduces or suppresses or regulate NOX/NADPH oxidase (particularly Nox1) activity, function or level.Described method comprises the Nox1 inhibitor peptide described herein giving described experimenter's effective dose, and wherein said peptide is optionally conjugated to or is connected to the reagent increasing described peptide and accumulate in cell.
The present invention is also provided in experimenter in need the method reducing reactive oxygen species (ROS) level or inhibit activities oxygen clusters (ROS) and produce, it comprises the Nox1 inhibitor peptide described herein giving described experimenter's effective dose, and described peptide is optionally conjugated to or is connected to the reagent increasing described peptide and accumulate in cell.
Described peptide is optionally conjugated to or is connected to the reagent increasing described peptide and accumulate in cell.Described be applicable to puting together of reagent described peptide will be made easier through film, described reagent such as can the carrier of permeates cell membranes.Described reagent can be can the carrier of permeates cell membranes, such as, be rich in the amino acid whose peptide of lotus positive electricity, such as, be rich in arginic peptide.Such as, being rich in arginic peptide can be the poly polyarginine-tag with sequence RRRRRRRRR (SEQ ID NO:46).Other cell-penetrating peptides can be selected from: derived from the NGR peptide (CNGRCG:SEQ ID NO:47) of aminopeptidase (CD13) N part, feeler foot leader peptide (Antennapedia Leaderpeptide) (CT) (KKWKMRRNQFWVKVQRG:SEQ ID NO:48), Bcl-2 binding peptide (caprinoyl-KNLWAAQRYGRELRRMSDEFEGSFKGL:SEQ ID NO:49), tat sequence (RKKRRQRRR:SEQ ID NO:50), Bufo siccus antibacterial peptide (buforin) (TRSSRAGLQFPVGRVHRLLRK:SEQ ID NO:51), the fragments of peptides (TRRQRTRRARRNR:SEQ ID NO:52) of human T lymphotrophic virus (HTLV) II type Rex, adipose membrane translocating peptides (KKAAAVLLPVLLAAP:SEQ ID NO:53), the NRP-1 targeting peptides (RPARPAR:SEQ IDNO:54) of streptomyces hygroscopicus (Streptomyces hygroscopicus) and penetrate element (penetratin) (RQIKIWFQNRRMKWKKGG:SEQID NO:55).
Peptide of the present invention can be used for preventing and/or treating any nadph oxidase relevant disease, it can comprise cardiovascular disease and disease, includes but not limited to: hypertension, atherosclerosis, cardiac hypertrophy, heart failure, myocardial infarction, restenosis, diabetes, diabetic nephropathy, intestinal mucosal injury (particularly the mucosa injury of inflammatory bowel), neointimal hyperplasia (neointimalhyperplasia) and angina pectoris; The peroxide that other and NADPH enzyme mediate produces and increases simultaneous disease and disease, includes but not limited to: arthritis, chronic inflammatory bowel disease (IBD), septicemia and other inflammatory diseasess, bronchial asthma, chronic obstructive pulmonary disease (COPD), lung infectious disease, pulmonary fibrosis, adult respiratory distress syndrome (ARDS), cancer, autoimmune disease, reperfusion injury, nephropathy, apoplexy, Alzheimer, parkinson disease and other neurodegenerative diseases, cystic fibrosis, organ rejection and pulmonary hypertension.In the broadest sense, peptide of the present invention can be used for preventing or treat the inflammatory diseases that any reactive oxygen species (ROS) mediates, include but not limited to: the disease of mediated phosphorylation, the disease of polymorphonuclear leukocyte mediation, macrophage-mediated disease, the disease of lipopolysaccharide mediate, the disease of tumor necrosis factor-alpha mediation, the disease that cytokine IFN-γ mediates, the disease of interleukin II mediation, inflammatory arthritis, triperchromic acid potassium arthritis (potassium peroxochromatearthritis), rheumatoid arthritis, osteoarthritis or Alzheimer.In preferred embodiments, the present invention relates to treatment or prevention, wherein nadph oxidase 1 (Nox1) activity or ROS active causing are selected from following disease: cardiovascular disease, respiratory system disease, metabolic disease, dermatosis, osteopathia, CNS disease, neuritic and/or neurodegenerative disease, kidney diaseases, sleep disorder, reproductive disease, affect eyes and/or lenticular disease, inflammatory diseases, hepatopathy, pain, cancer, tumor, acute renal failure (ARF), anaphylactic disease, dermatosis, UV damages, the dermatosis caused by aging, the photic damage of skin, the skin injury of ROS induction, cross presenility, diseases associated with senescence or disease, gastrointestinal system disorder, blood vessel generation disease, atherosclerosis, restenosis after support inserts and/or hypertension etc.
In preferred embodiments, the invention provides the method for inhibition tumor cell growth in the experimenter needed, described method comprises the Nox1 inhibitor peptide of the present invention, its analog or the examples of conservative variations that give to treat effective dose.Described peptide is optionally conjugated to or is connected to the reagent increasing described peptide and accumulate in cell.In the most preferred embodiment, the suppression of described growth of tumour cell is the suppression grown the keratinocyte of tumorigenesis.In another preferred embodiment of the present, described for treating and/or preventing the method that cutaneous tumor occurs after long-term UV (UVA or UVB) exposure.
In in preferred, method of the present invention is applicable to multiplely be selected from following disease: the restenosis after the prevention of the skin injury of cancer, ROS induction, skin aging and other diseases associated with senescence, atherosclerosis, support insert and/or hypertension etc.In highly preferred embodiment, the cancer for the treatment of by method of the present invention is selected from skin carcinoma, cancerate before skin injury, colon cancer, adenocarcinoma of colon, carcinoma of prostate, optimum and malignant papilloma etc.Described cancer can be melanoma or hematologic malignancies, such as acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS) or myeloid leukemia, such as chronic myeloid leukemia (CML) or acute myeloid leukemia (AML).Described cancer can be cancer of pancreas.
Described cancer can be the carcinogenecity cell transformation of Ras induction, such as colorectal cancer.Described cancer can be the gastrointestinal tumor of adenocarcinoma or helicobacter pylori infections induction.Described cancer can be caused by human papillomavirus.Described cancer can be caused by xeroderma pigmentosum C type (XPC) gene silencing.
Broadly, the present invention includes beauty treatment or pharmaceutical composition, it comprises cosmetic or medicine or the treatment at least one peptide of the present invention of effective dose or the mixture of peptide of the present invention and pharmaceutically acceptable salt, and generally includes at least one cosmetically or pharmaceutically acceptable excipient (excipient) or adjuvant.
Present invention also offers medicine or the alimentation composition of suppression or adjustment Nox1 activity in experimenter, it comprises peptide of the present invention.In other preferred embodiments, the invention provides selectivity in the experimenter needing it reduce and/or suppress the compositions of Nox1 activity, or the compositions that selectivity reduction reactive oxygen species (ROS) level or inhibit activities oxygen clusters (ROS) produce in the experimenter needing it, wherein said compositions includes the Nox1 inhibitor peptide of the present invention of effective amount.In the most preferred embodiment, the invention provides and reduce and/or suppression Nox1 activity for selectivity in the experimenter needing it, and/or reduce the compositions that reactive oxygen species (ROS) level or inhibit activities oxygen clusters (ROS) produce, the peptide of the aminoacid sequence that it includes having of effective amount and is selected from SEQ ID NO:1 to 42---preferably SEQ ID NO:1 to 19---, its analog or examples of conservative variations, wherein said peptide is optionally conjugated to or is connected to the reagent increasing described peptide and accumulate in cell.
In another, the present invention relates to the compositions comprising peptide of the present invention, wherein said compositions also comprises pharmaceutically acceptable excipient (excipient) or carrier.Particularly, the present invention relates to Nox1 inhibitor peptide of the present invention as active component, the pharmaceutically acceptable carrier of at least one, excipient (excipient) and/or diluent, for the preparation of the purposes of pharmaceutical composition treating and/or preventing disease disclosed herein.Described pharmaceutical composition comprises Nox1 inhibitor peptide, and the pharmaceutically acceptable carrier of at least one, excipient (excipient), binding agent, disintegrating agent, fluidizer (glident), diluent, lubricant, coloring agent, sweeting agent, flavoring agent, antiseptic etc.Pharmaceutical composition of the present invention, by known way, is prepared under the dosage level be applicable in the solid of routine or liquid-carrier or diluent and conventional medication level adjuvant.Form of medication comprises, such as, and pill, tablet, thin film tablet, coated tablet, capsule, Liposomal formulation, micron preparation and nanometer formulation, powder and precipitate (deposit).In addition, the present invention also comprises the pharmaceutical preparation used for parenteral, comprise skin, intradermal, gastric, Intradermal, Ink vessel transfusing, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, in cheek, percutaneous, rectum, subcutaneous, Sublingual, local or applied dermally, described goods also comprise new type of peptides of the present invention except conventional excipients (vehicle) and/or diluent.Can see " Remington'sPharmaceutical Sciences " Mack Publishing Co., Easton PA for the preparation of peptide of the present invention and the technology of administration.
Peptide of the present invention can also give with the form of its pharmaceutically active salt.The pharmaceutically active salt be applicable to comprises acid-addition salts and alkali metal or alkali salt.Such as, the salt of sodium, potassium, lithium, magnesium or calcium can be obtained.Nox1 inhibitor peptide of the present invention or peptide combinations and organic acid and mineral acid form pharmaceutically acceptable salt.The example of acid be applicable to formed for described acid-addition salts is hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, para-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acids, perchloric acid, nitric acid, formic acid, propanoic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, acetone acid, phenylacetic acid, benzoic acid, para-amino benzoic acid, P-hydroxybenzoic acid, methanesulfonic acid, ethyl sulfonic acid, nitrous acid, ethylenehydrinsulfonic acid, ethionic acid (ethylenesulfonicacid), p-methyl benzenesulfonic acid, LOMAR PWA EINECS 246-676-2, p-anilinesulfonic acid., camphorsulfonic acid, china acid, mandelic acid, adjacent methyl mandelic acid, hydrogen-benzenesulfonic acid, picric acid, fatty acid, D-o-tolyl tartaric acid, hydroxymalonic acid., α-toluic acid, (adjacent, between, right) toluic acid, naphthylamine sulfonic acid, and other mineral acid known in those skilled in the art or carboxylic acids.Described salt by by free alkali with enough needed for acid contact thus generate salt in a usual manner and prepare.
In an embodiment of this aspect of the invention, Nox1 inhibitor peptide of the present invention can with non-peptide moiety conjugation.Treat with the polymer molecule of described peptide coupling it can is any applicable polymer molecule, such as natural or synthesis homopolymer or heteropolymer, its common molecular weight ranges is about 300-100000Da, such as about 500-20000Da.The example of polymer molecule be applicable to comprises and is selected from following polymer molecule: polyalkylene oxide (polyalkylene oxide, PAO) (ployalkylene glycol (PAG) is comprised, such as Polyethylene Glycol (PEG) and polypropylene glycol (PPG), ramiform PEG, polyvinyl alcohol (PVA)), polycarboxylic acids, polyvinylpyrrolidone, polyethylene-altogether-maleic anhydride, polystyrene-altogether-maleic anhydride, glucosan (comprising Sensor Chip CM 5), or any other is suitable for the biopolymer reducing immunogenicity and/or increase Functional in vivo half-life and/or serum half-life.Another example of polymer molecule is human albumin or another kind of abundant plasma protein.The polymer that ployalkylene glycol derives is normally biocompatible, nontoxic, no antigen, non-immunogenicity, has a multiple water solubility properties and is easy to secrete from the organism of living.
Also can join in drug delivery system and/or slow-released system by Nox1 inhibitor peptide of the present invention, term " delivery system " relates to the diluent, adjuvant, excipient (excipient) or the carrier that give together with peptide of the present invention.These beauty treatments or pharmaceutical carrier can be liquid, such as water, oil or surfactant, comprise oil, animal, plant or synthesis source those, such as but not limited to Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Oleum sesami, Oleum Ricini, polysorbate, Isosorbide Dinitrate, ether sulfuric ester (ether sulfate), sulfuric ester, betanin, glucosides, maltoside, fatty alcohol, nonoxynolum, poloxamer, polyoxyethylene, Polyethylene Glycol, dextrose, glycerol, digitonin and analog.In " the Remington's Pharmaceutical Sciences " of E.W.Martin, describing diluent, adjuvant or excipient is suitable carrier.Term " slow release " uses with the conventional sense relating to the delivery system of compound, and it provides the release gradually of this compound within a period of time, preferably, although and not necessarily, keep the compound emission levels of relative constancy within a period of time.To send or the example of slow-released system is liposome, Coliposomes, elaiosome, lipoid plastid (niosome), containing alcohol liposome (ethosome), millimeter particle (milliparticle), micron particle (microparticle), nanoparticle and solid lipid nanoparticle, the lipid carrier of nanostructured, sponge, cyclodextrin, vesicle (vesicle), micelle, the mixed micelle of surfactant, the micelle of surfactant-phospholipid mixing, millimeter ball (millisphere), micron ball (microsphere) and nanosphere, fat spheroid (liposphere), millimeter capsule (millicapsule), micron capsule (microcapsule) and Nano capsule, and microemulsion (micro-emulsion) and nano-emulsion, can be added them to realize the larger permeability of Nox1 inhibitor peptide and/or to improve its pharmacokinetics and pharmacodynamic properties.Preferably to send or slow-released system is liposome, the micelle of surfactant-phospholipid mixing and microemulsion, more preferably there is the water-in-oil microemulsion of the internal structure of reverse micelle.
When compositions of the present invention is used as bright protective agent or defying age reagent, it also comprises conventional cosmetic additive and adjuvant, particularly be selected from lipid (fatty substance), organic solvent, thickening agent, softening agent, antioxidant, opacifier, stabilizing agent, isostearyl glyceryl pentaerythrityl ether (emollient), hydroxy acid, defoamer, wetting agent, vitamin, spice (fragrance), antiseptic, surfactant, filler, sequestering agent (sequestering agent), polymer, propellant, alkalization or acidulant, dyestuff, coloring agent or be usually mixed with any other compositions of cosmetics, especially for the antisum/sunscreen composition preparing Emulsion form.
Method and composition of the present invention can use with other compositionss and methods combining being used for the treatment of disease.Such as, conventionally can adopt operation, radiation, chemotherapy or immunotherapy, treat tumor in conjunction with method and composition of the present invention.Then, after this can give patient to extend the dormancy of small transfer by method and composition of the present invention, and stablize and suppressing the growth of any residual primary tumor.Also can by compositions of the present invention and other anti-angiogentic compound or protein, fragment, antiserum, receptor stimulating agent, the receptor antagonist of other anti-angiogenic protein matter combine.In addition, novel Nox1 inhibitor peptide of the present invention can combine with pharmaceutically acceptable excipient (excipient) and optional sustained-release matrix (such as, biodegradable polymer), to form therapeutic combination.
In addition, compositions of the present invention can give with other treatment simultaneously, such as, carries out with chemotherapy or radiation treatment plan simultaneously.The example of the chemotherapeutant that can be combined with novel Nox1 inhibitor peptide of the present invention comprises alkylating agent, such as chlormethine, ethyleneimine compounds, alkylsulfonate and other there is the compound of alkylating, such as nitroso ureas, cisplatin, dacarbazine; Antimetabolite, such as folic acid, purine or Pyrimidine antagonists; Mitotic inhibitor, such as vinca alkaloids and podophyllotoxin derivative; Cytotoxic antibiotics and camptothecin derivative.Preferred chemotherapeutant or chemotherapy comprise amifostine (ethyol), cisplatin and/or other platinum compounds (preferably including carboplatin and/or oxaliplatin), dacarbazine (DTIC), dactinomycin, chlormethine (chlormethine), streptozotocin, cyclophosphamide, carmustine (carrnustine) (BCNU), lomustine (CCNU), doxorubicin (amycin), Mycocet (Doxil (doxil)), gemcitabine (gemzar), daunorubicin, daunorubicin liposome (DaunoXome), procarbazine, mitomycin, cytosine arabinoside, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecine, CPT-11, 10-hydroxyl-7-Ethyl-camptothecin (SN38), dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon-ALPHA, interferon beta, irinotecan, mitoxantrone, topotecan, leuprorelin, megestrol, melphalan, purinethol, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozotocin, tamoxifen, teniposide, testolactone, thioguanine, phosphinothioylidynetrisaziridine, uracil mustard, vinorelbine, chlorambucil, and their combination.
When be used for the treatment of or process atherosclerosis, support insert after restenosis and/or hypertension time, can be used alone Nox1 inhibitor peptide of the present invention or be used for the treatment of and/or process atherosclerosis, support insert after restenosis and/or hypertensive other drug be combined.Described other drug can include but not limited to thrombolytic agent (such as streptokinase), tissue plasminogen activator, fibrinolysin and urokinase, antithrombotic agents (such as tissue factor protein enzyme inhibitor (TFPI)), the anticoagulant protein (NAPs) that extracts from nematicide etc., inhibitors of metalloproteinase, antiinflammatory, antidiabetic or antihyperglycemic agents (comprising insulin, Insulin secretagogues or insulin sensitizers), SGLT-2 inhibitor, AT 1receptor antagonist, HMG-CoA reductase inhibitor, Angiotensin-Converting (ACE) inhibitor, calcium channel blocker, aldosterone synthase inhibitors, aldosterone antagonists, Angiotensin-Converting/neutral endopeptidase (ACE/NEP) double inhibitor, endothelin antagonist, diuretic etc.
When be used as antidotal agent or bright protective agent time, Nox1 inhibitor peptide of the present invention can with following one or more combine: plant/plant extract; Thiodipropionic acid (TDPA) and ester thereof; Retinoic acid (such as, all-trans vitamin A acid, 9-cis-retinoic acid, phytanic acid and other); Hydroxy acid (comprising alpha-hydroxy acid and β-hydroxy acid), salicylic acid and Salicylate; Exfoliator (such as, glycolic, 3,6,9-trioxa heneicosanedioic acids etc.); Estrogen synthesis enzyme stimulus compound (such as, caffeine and derivant); The compound (such as, linolenic acid, linoleic acid, finasteride and composition thereof) of 5 alpha-reductase activity can be suppressed; Barrier function enhancing agent (such as, ceramide, glyceride, cholesterol and ester thereof, Alpha-hydroxy and ω-hydroxy fatty acid and ester etc. thereof); Collagenase inhibitors; And elastase inhibitor etc.
Described defying age or light protection group compound also can comprise one or more following reagent: plump dose of Percutaneous absorption enhancer, isostearyl glyceryl pentaerythrityl ether, skin (skin plumper), optical diffusant (opticaldiffuser), sunscreen, exfoliator and antioxidant.Isostearyl glyceryl pentaerythrityl ether provides the function benefit strengthening skin smoothness and reduce microgroove and coarse wrinkles appearance.These comprise, such as, and isopropyl myristate, vaseline, isopropyl lanolate, silicone (such as, methyl-silicone oil, dimethicone), oil, mineral oil, fatty acid ester, or its any mixture.Preferably, isostearyl glyceryl pentaerythrityl ether accounts for about 0.1% weight of composition total weight to about 50% weight.
Plump dose of skin is as the collagen protein reinforcing agent of skin.To be applicable to and the example of preferred plump dose of skin is palmitoyl oligopeptide.Plump dose of other skins are collagen protein and/or other glycosaminoglycans (GAG) reinforcing agent.When there is plump dose of described skin, it can account for about 0.1% weight of described composition total weight to about 20% weight.Optical diffusant is change skin surface optometry (optometrics), causes the dimness of vision of such as microgroove and wrinkle and softening granule.The example that can be used for the optical diffusant in the present invention includes but not limited to boron nitride, Muscovitum, nylon, poly-methyl first acrylates (PMMA), polyurethane powder, sericite, silicon dioxide, silicone powder, Talcum, polytetrafluoroethylene, titanium dioxide, zinc oxide, or its any mixture.When there is described optical diffusant, it can account for about 0.01% weight of described composition total weight to about 20% weight.
Also can comprise protection skin and avoid damaging ultraviolet sunscreen.Preferred sunscreen is to provide those of the protection of UVB and UVA widely, such as octocrylene, avobenzone (Parsol 1789), octyl methoxycinnamate, ethylhexyl salicylate, oxybenzone, homosalate (homosylate), benzophenone, camphor derivatives, zinc oxide and titanium dioxide.When there is described sunscreen, it can account for about 0.01% weight of described compositions to about 70% weight.
The exfoliator be applicable to comprises, such as, and alpha-hydroxy acid, β-hydroxy acid, oxyacid (oxaacid), containing oxygen diacid (oxadiacid), and its derivant such as its ester, acid anhydride and salt.The hydroxy acid be applicable to especially comprises glycolic, lactic acid, malic acid, tartaric acid, citric acid, 2-hydroxy alkanoic acid, mandelic acid, salicylic acid and derivant thereof.Preferred exfoliator is glycolic.When there is described exfoliator, it can account for about 0.1% weight of described compositions to about 80% weight.
At other on, the Main Function of antioxidant be from skin scavenging free radicals to protect the skin person that resists environmental attack.In compositions of the present invention, the example of spendable antioxidant comprises the compound with phenolic hydroxyl group functional group, such as ascorbic acid and derivant/ester thereof, beta-carotene, catechin, curcumin, ferulic acid derivative are (such as, ferulic acid ethylester, sodium ferulate), gallic acid-derivate (such as, propyl gallate), lycopene, 2-cyclopenten-2,3-diol-1-one., rosmarinic acid, tannic acid, tetrahydrocurcumin, tocopherol and derivant thereof, uric acid, or its any mixture.Other antioxidants be applicable to are those (reduction or the non-reduced forms) with one or more mercapto functional group (-SH), such as glutathion, thioctic acid, TGA and other sulfhydryl compounds.Antioxidant can be inorganic matter, such as bisulfites, metabisulfite, sulphite or other comprise inorganic salt and the acid of sulfur.
The additive of other routines comprises vitamin, such as tocopherol and ascorbic acid; Vitamin derivative, such as ascorbic acid monopalmitate; Thickening agent, such as hydroxy alkyl cellulose; Gellant; Structure agent (structuring agent), such as bentonite, Montmorillonitum, aluminium-magnesium silicate and Lithium metasilicate magnesium; Metal-chelating reagent, such as EDTA; Pigment, such as zinc oxide and titanium dioxide; Coloring agent; Isostearyl glyceryl pentaerythrityl ether; And wetting agent.
The present invention also extends to part or the fragment of Nox1 gene or its mRNA, particularly the long variant of nadph oxidase 1 isoform, nadph oxidase activator 1 isoform 3, nadph oxidase organizator (organizer) 1 isoform.The present invention be more particularly directed to encode the nucleotide sequence of peptide of the present invention." part or fragment " is defined as at least about 8 nucleotide or a preferred about 12-17 nucleotide or more preferably at least about the minimum length of 18-25 nucleotide and the greatest length had at least about 5000 nucleotide.Also can use the genome equivalents (equivalent) more than 5000 nucleotide.This definition comprises all length in 8-5000 nucleotide range.Therefore, this definition comprises the nucleic acid of at least 12,15,20,25,40,60,80,100,200,300,400,500 or 1000 nucleotide, or have any number in these numerical value nucleotide nucleic acid (such as, 13,16,23,30,28,50,72,121 nucleotide etc.), or there is the nucleic acid more than the nucleotide of any number between 500 nucleotide or 500.The present invention includes all novel nucleic acids, its complementary nucleic acid or function equivalence nucleic acid, described novel nucleic acids its there is the nucleotide that at least 8 are derived from the nucleic acid of Nox1 inhibitor peptide of the present invention of encoding.In the most preferred embodiment, the invention provides the nucleic acid of the peptide of coding SEQ ID NO:2 to 42, its analog or examples of conservative variations.In addition, the present invention relates to plasmid or the carrier of the nucleic acid comprising Nox1 inhibitor peptide of the present invention of encoding.Any disease mentioned by nucleic acid of the present invention, plasmid and carrier can be used for treating herein.
One or more detectable can be adopted to carry out labelling peptide of the present invention.Described label directly or by joint can be conjugated to described peptide.When peptide of the present invention and direct label are puted together, described direct label is detectable entity under native state suitably.Such as, when described direct label be coloured particle (such as dye sols (dye sol), metal-sol (metallic sol) (such as, gold colloidal)) and colored latex granule time, its can be bore hole visible or optical filter (optical filter) auxiliary under be visible.When described direct label is fluorescent marker, can make its accept apply stimulation (such as, ultraviolet light) to promote fluorescence.The detectable substance be applicable to comprises multiple enzyme, prothetic group, fluorescent material, luminescent material and active material.
In one embodiment of the invention, Nox1 inhibitor peptide of the present invention can be conjugated to one or more detections, diagnosis or therapeutic agent and be delivered to disease location (cancerous cell in such as cancerous cell, particularly human body) with selectivity.Described put together can be direct or by use joint, be similar to above-mentioned sewing.Corresponding disease detection, Diagnosis and Treat method are corresponding aspects of the present invention, as the described peptide in described detection, Diagnosis and Treat method.Detection, Diagnosis and Treat agent can be naturally occurring, modified or synthesis.Therapeutic agent can promote or suppress any bioprocess involved in human disease's path.Use is conjugated to one or more detections, diagnosis or therapeutic agent and is delivered to the peptide of the present invention of disease location with selectivity, described detection, diagnosis and therapy can be implemented in vitro, particularly implement at the sample obtained from experimenter, or implement in the body of experimenter (patient) that is to be tested or that treat.Such as, other reagent can be the recognized in the art therapeutic agents being applicable to Therapeutic cancer.Specific binding between described peptide and cell interacts and enables described therapeutic agent by the accurately cancerous cell of targeting in mammalian patient.The therapeutic agent being applicable to this purposes can comprise cell death inducing, cell death, cell differentiation, cells arrest (cell stasis) and/or anti-angiogenic generation, or affects any compound of cancer cell survival and/or growth rate.
The present invention also provides the method being prepared Nox1 inhibitor peptide of the present invention by solid phase or liquid phase synthesis, and wherein said method optionally comprises and the step increasing the reagent that accumulates in cell of described peptide and put together or be connected.
Delivering method
Once be made into preparation, multiple known approach and technology can be used compositions of the present invention (comprising peptide of the present invention or polynucleotide) to be delivered in subject.Such as, compositions can be used as Injectable solution, suspension or Emulsion form and provides, and by using conventional syringe needle and syringe or using the parenteral of liquid injection system (liquid jet injection system), subcutaneous, epidermis, intradermal, intramuscular, intra-arterial, intraperitoneal, intravenous injection to give.Also compositions local can be given to skin or mucosal tissue, such as carry out per nasal, tracheal strips, through intestinal, rectum or vagina administration, or to be applicable to breathe or the finely divided spray form of lung administration provides.Other administering modes comprise oral administration, suppository, sublingual administration, and initiatively or passive transdermal delivery technology.
Send scheme
Described peptide/polynucleotide is given by above-mentioned any applicable method.Determine the suitable amount of described peptide by experience, but be usually located in scope given below.The single-dose of often kind of peptide is enough to produce beneficial effect to patient, such as, but should be appreciated that and exceed that once to give described peptide be useful, usual dosage regimen in this case can be, weekly or twice in 2-4 week of every 6 months, or in one week of every 4-6 month once a day.Should be appreciated that, can by often kind of peptide or polynucleotide, or the compositions of peptide and/or polynucleotide gives patient either individually or in combination.
Dosage depends on many factors, comprises the character of described compositions, route of administration, and the timetable of dosage regimen and opportunity.The applicable dosage of the combination of molecule of the present invention or molecule can be following order: each administration 1 μ g is also up to 10 μ g, is up to 15 μ g, is up to 20 μ g, is up to 25 μ g, is up to 30 μ g, is up to 35 μ g, is up to 50 μ g, is up to 100 μ g, is up to 500 μ g or more.The dosage be applicable to can be less than 15 μ g, but is at least 1ng or at least 2ng or at least 5ng or at least 50ng or at least 100ng or at least 500ng or at least 1 μ g or at least 10 μ g.For molecules more of the present invention, the dosage used can be higher, such as, be up to 1mg, be up to 2mg, be up to 3mg, be up to 4mg, be up to 5mg or higher.Described dosage can liquid preparation form be applicable to concentration provide, to allow to be suitable for the volume with selected administration.
embodiment
Test other two kinds of peptide A and B of the present invention, and compared with control peptide C, listed by table 5 below.
Peptide A (SEQ ID NO:41) is included in the tat sequence of sequence SEQ ID NO:20N end.Peptide B (SEQ ID NO:42) is included in the tat sequence of sequence SEQ ID NO:21N end.PEPC (SEQ ID NO:43) is control peptide (or scrambled peptide).
Table 5: the target of peptide for inhibiting and sequence
Peptide The target of peptide Sequence
A SH3-NoxA1 tat-P-P-T-V-P-T-R-P-S(SEQ ID No:41)
B SH3-NoxA1 tat-I-Q-S-R-C-C-T-V-T(SEQ ID No:42)
C Out of order tat-C-L-R-I-T-R-Q-S-R(SEQ ID No:43)
First trypan blue exclusion mensuration and MTT is used to be determined at the cytotoxic effect normal primary Human keratinocytes being tested often kind of peptide.As shown in Figure 1A and 1B, significant toxicity is not observed after employing is less than the peptide process of 50 μ Μ.
Find that Nox1 inhibitor peptide reduces intracellular ROS level effectively, as following result prove.
Use the ROS level (Fig. 2 A) in the primary Human keratinocytes of CM-H2DCF-DA probe assay employing 10 or 50 μ Μ peptide A, B and C process 24h.The remarkable decline of ROS level is observed in the cell adopting Nox1 inhibitor peptide A and B process.In order to check the specificity of these peptides, in human colon adenocarcinoma HT29 cell line, assess ROS level, in described cell line, Nox1 is the sole active member in Nox family.In HT29 cell line, the measurement of ROS level shows, adopts the process of Nox1 inhibitor peptide A and B to cause the remarkable reduction of ROS level, shows the specific inhibitory effect (Fig. 2 B) of these peptides to Nox1 activity.
In order to check the specificity of these peptides, using the shRNA for the lentivirus mediated of Nox1 and Nox2 to express and suppressing endogenous Nox1 and Nox2 protein expression.ShNox1 and shNox2 stably suppresses Nox1 and Nox2 of more than 80% to express (Fig. 3 A) respectively.Then the keratinocyte of peptide A process shCtrl, shNox1 or shNox2 transduction is adopted.The measurement of ROS level discloses ROS steady-state level in the cell adopting the process of peptide A transduce to shNox1 and does not act on, and therefore proves that peptide A is with the effectiveness of very high (close to 100%) and dependent ROS generation (Fig. 3 B) of specific inhibition Nox1.In addition, in the cell adopted or do not adopt shCtrl and shNox1 of peptide A process to transduce, nadph oxidase activity (Fig. 3 C) is measured.Find that nadph oxidase is active at the cell of shNox1 transduction, the cell of peptide A process and adopting in the cell of the shNox1 of peptide A process transduction to reduce, because herein illustrating effectiveness and the specificity of peptide A blocking-up Nox1 activation equally.
Assessment in the body of embodiment 1---Nox1 inhibitor peptide
In order to assess the photoprotection of Nox1 inhibitor peptide A, this peptide is first checked whether to generate inhibited (Fig. 4) to the ROS of Nox1 induction in mice and Human keratinocytes.Result shows, in mice and Human keratinocytes, peptide A generates the ROS that Nox1 induces and Nox activity has similar inhibitory action.
In order to check peptide A to the effect of Nox1 activity in mouse skin, adopt the peptide A Local treatment SKH-1 mice (Fig. 5 A) of various dose.Result shows, when giving peptide A with 3 and 12mg/kg, peptide A effectively suppresses Nox activity to reach 3 days most.Then test peptides A is to the effect (Fig. 5 B) of the mice that UVB irradiates.Result shows, pre-irradiation 10 minutes adopt 3 and the Local treatment of the 12mg/kg peptide A Nox that effectively blocks UVB induction in mice activate.
In order to check the photoprotection of peptide A; the effect that the squamous cell carcinoma (SCC) using SKH-1 hairless mouse measurement peptide A administration to induce UVB is induced, described SKH-1 hairless mouse is the well-defined mouse model for studying the photic cancer that cutaneous tumor (comprising optimum papilloma and pernicious SCC) occurs.The effectiveness of this model is described by the remarkable similarity in the carcinogenesis of UVB induction in these mices and application on human skin between UV carcinogenesis path.SKH-1 hairless mouse creates very valuable data in the dosage occurred for the cutaneous tumor after Long-term UVB irradiation, time course and action spectrum.In this research, give weekly peptide A (3mg/kg) 3 times, and within 10 minutes, gave before each UVB exposes.Result shows, adopts the process of peptide A to make tumor quantity and tumor size reduce by 2.3 and 2.9 times (Fig. 6 A and 6B) respectively.In addition, in the mice of peptide A process, the gross tumor volume of 57% is less than 5mm 3, by contrast, adopt in the mice of control peptide C process and only have the gross tumor volume of 8% to be less than 5mm 3(Fig. 6 C).
In mouse tumor, the measurement of nadph oxidase activity shows that peptide A is suppressing the remarkable effectiveness (Fig. 7) in Nox activity.Compared with the mice processed with excipient (vehicle), from the mice of peptide A process, in isolated non-lotus tumor skin and tumor, nadph oxidase activity reduces by 70% and 54% (Fig. 7) respectively.
embodiment 2---the effect of Nox1 inhibitor peptide in treatment of cancer
Table 6
embodiment 2.1: to the interaction in vitro of cancerous cell line
The different cell lines listed in table 6 are assessed the effect that Nox1 suppresses.Cell proliferation, cell cycle progress and apoptosis between the cell of more untreated cell and the process of employing Nox1 inhibitor peptide.
embodiment 2.2: cell is to the xenotransplantation of NOD/SCID mice
Trypsin treatment is carried out to cell, rinses in DPBS, and be again suspended in the DPBS containing 25% matrigel (BD Biosciences).By 2 × 10 6individual cell subcutaneous injection is in 6-8 NOD/SCID mice in age in week (The Jackson Laboratory).For often kind of cell line, mice is divided into two groups: one group and processes with excipient (vehicle), the Nox1 inhibitor peptide process of another group.Formed and tumor growth in the monitoring in time tumor reaching most 2 months.Often kind of tumor dissected, measures, be fixed in 4% paraformaldehyde, be embedded in paraffin, and process is dyeed for H & E.The assessment rate of increase and apoptosis rate.
embodiment 2.3: mouse model
Use following model measurement Nox1 inhibitor peptide to the effect of cutaneous tumor: Patch +/-the BCC of spontaneity and UVB induction in mice; The SCC of UVB induction in SKH-1 mice; In BRAF mice, spontaneous melanoma is formed.For often kind of model, mice is divided into two groups: one group and processes with excipient (vehicle), the Nox1 inhibitor peptide process of another group.
embodiment 3---Nox1 inhibitor peptide is to the effect of inflammatory and autoimmune disease
Table 7
embodiment 3.1: interaction in vitro
Arthritis detects Nox1 inhibitor peptide, activates the macrophage from peripheral blood lymphocyte (PBL) when Nox1 inhibitor peptide exists and do not exist.Detect the secretion of different inflammatory mediator (TNF-a, IL-6, IL-1 β, IL-8, IL-10).
When there is and do not exist Nox1 inhibitor peptide by assessing, the inflammatory reaction of IgE-DNP induction tests Nox1 inhibitor peptide to the effect of asthma.
embodiment 3.2: effect in body
Adopt the arthritic rat model of Nox1 inhibitor peptide process of various dose to assess the improvement of peptide to clinical score and the prevention of bone destruction.
In addition, the effectiveness of test Nox1 inhibitor peptide in asthma and rat ears allergy when there is and do not exist Nox1 inhibitor peptide.
embodiment 4---the effect that Nox1 inhibitor peptide occurs blood vessel
Table 8
For the interaction in vitro that assessment Nox1 inhibitor peptide occurs normal blood vessels, test the endothelial cell migration and tubular structure formation that use HUVEC.
Act on in the body that assessment Nox1 inhibitor peptide occurs normal blood vessels, inspection adopts the skin heart generation in C57BL/6 and the SKH-1 mouse model of excipient (vehicle) or the process of Nox1 inhibitor peptide.
For the effect that assessment Nox1 inhibitor peptide occurs tumor vessel, matrigel blood vessel can be used to occur to measure and heteroplastic transplantation model.Mice is injected by various kinds of cell system listed in table 6, between employing excipient (vehicle) and the mice of Nox1 inhibitor peptide process, then compares blood vessel occur.
embodiment 5---Nox1 inhibitor peptide is to the effect of angiopathy and cardiovascular disease
Table 9
The effect that test Nox1 inhibitor peptide is to hemangioma when there is and do not exist Nox1 inhibitor peptide---the endotheliocyte conversion undertaken by T antigen (PymT) in the middle of murine polyomavirus---.
The effect that assessment Nox1 inhibitor peptide is to hypertension when there is and do not exist Nox1 inhibitor peptide---the hypertension induction after infusion Angiotensin II---.
embodiment 6---Nox1 inhibitor peptide is to the effect of neurodegenerative disease
Table 10
For assessment Nox1 inhibitor peptide is to the effect of parkinson disease and Alzheimer, use the neuronal cell derived from Skin Cell by iPS technology.Be also tested for the effect of Nox1 inhibitor peptide to ROS level in the neuronal cell from patient and healthy skin.
The effect of---mouse model of experimental autoimmune encephalomyelitis (EAE)---to multiple sclerosis of assessment Nox1 inhibitor peptide.In fact the EAE model of PLP139-151 inducing peptide in SJL mice is employed.
Embodiment 7---Nox1 inhibitor is to the effect of dermatosis
Table 11
The atopic dermatitis of assessment Nox1 inhibitor peptide to the keratinocyte by IgE induced activation, the effect of inflammatory mediator secretion when there is and do not exist Nox1 inhibitor peptide.Be also tested for the ear allergy of the IgE induction when there is and do not exist Nox1 inhibitor peptide.
For assessment Nox1 inhibitor peptide is to leucoderma wind action, the epidermis of reconstruction and primary melanocyte can be used to test melanocyte desorption.
Finally, the epidermis of primary culture keratinocytes and reconstruction tests the anti-aging effects of Nox1 inhibitor peptide.Differentiation and old and feeble label is compared between the cell adopting excipient (vehicle) or the process of Nox1 inhibitor peptide.
Embodiment 8---Nox1 inhibitor peptide is to the effect of skin aging
For the anti-aging effects of assessment Nox1 inhibitor peptide A (SEQ ID NO:41), the acceleration first assessed in XPC deficient mice is old and feeble.For this reason, sound at 4 monthly ages (youth) and 1.5 years old age (old) XPC and deficient mice (is respectively XPC + /+and XPC -/-) in have evaluated the expression (Fig. 9 A and 9B) of the truncate version presenilin of the nuclear lamina protein A relevant with progeria syndrome.Result shows, presenilin level raised with the age, and it is at XPC -/-expression ratio in mice is higher in wild-type mice.
For checking this phenotype whether relevant to the excessive activation of nadph oxidase, in mouse skin, first measure the activity (Figure 10 A) of nadph oxidase.Result shows, NOX activity raised with the age, and it is at XPC -/-higher in mice than in wild-type mice.Change because some research discloses mitochondrion and mitochondrial DNA (mtDNA) along with the wide spectrum of aging, what comprise structure of mitochondria disintegrates increase, the decline of Mitochondria (OXPHOS) function and the accumulation of mtDNA sudden change, afterwards at young and old XPC + /+and XPC -/-oXPHOS protein expression level (Figure 10 B) is compared between mice.Result shows, compared with the wild-type mice of youth, the expression of old wild-type mice mesocomplex I, II, III significantly reduces.In addition, these complexs are at the XPC of youth -/-expression in mice is the same with in old wild-type mice low, this and XPC -/-the presenility of crossing of mice is correlated with.
For research nadph oxidase is active in the effect of crossing presenility in XPC deficient mice, Nox1 inhibitor peptide or excipient (vehicle) has been adopted to process 1 monthly age XPC + /+and XPC -/-mice, processed weekly 3 times in 3 months.Then assess presenilin in Mus epidermis cell and express (Figure 11), OXPHOS protein expression (Figure 12) and metabolic characteristics.Result shows, the acceleration decreased in XPC deficient mice the suppression of Nox1 activity is old and feeble.In fact, Nox1 suppresses to have blocked the increase (Figure 11) of the protein expression of presenilin and stops the reduction (Figure 12) of the expression of OXPHOS complex I, II and III.In order to totally understand the effect that Nox1 inhibitor peptide strikes to XPC the metabolism change that low (knockdown) induces, employ proteomics method.Result shows, in pentose phosphate pathway, TCA circulation, mitochondrion OXPHOS and fatty acid beta oxidation, involved some protein expressions significantly reduce.Employing Nox1 inhibitor peptide process mice eliminates the effect that XPC defect changes metabolism.
Adopt the Nox1 inhibitor peptide as shown in SEQ ID NO:2 to carry out these test and obtain identical result.
These results demonstrate together, and Nox1 inhibitor peptide A and the Nox1 inhibitor peptide as shown in SEQ ID NO:2 can block the presenility excessively of XPC defect induction.

Claims (22)

1. length is 7 to 35 amino acid whose peptides, and it comprises a part for following general formula: X1-P-X2-X3-P-X4-R (SEQ ID NO:1), wherein
X1 is A, P or Q;
X2 is P, T, V, A, S, C, M or K;
X3 is L, I, V or A; And
X4 is T, V, S, A or M,
It produces increase relevant pathological conditions or the method for disease for preventing and/or treating and/or reactive oxygen species active to Nox1.
2. the peptide that uses of the method for claim 1, wherein said peptide or moiety ground are any following sequence in following table 1 or any following sequence that comprises in following table 1:
Table 1:
PPTVPTR(SEQ ID NO：2) PPSVPTR(SEQ ID NO：3) PPMVPTR(SEQ ID NO：4) PPCVPTR(SEQ ID NO：5) PPPVPTR(SEQ ID NO：6) PPAVPTR(SEQ ID NO：7) PPVVPTR(SEQ ID NO：8) PPTVPSR(SEQ ID NO：9) PPTVPMR(SEQ ID NO：10) PPTVPVR(SEQ ID NO：11) PPTVPAR(SEQ ID NO：12) QPTAPTR(SEQ ID NO：13) PPTLPTR(SEQ ID NO：14) PPTIPTR(SEQ ID NO：15) PPTAPTR(SEQ ID NO：16) PPKVPTR(SEQ ID NO：17) QPTVPTR(SEQ ID NO：18) APTVPTR(SEQ ID NO：19)
。
3. the peptide used any one of aforementioned claim, described peptide is conjugated to cell-penetrating peptides by covalent bond or non-covalent bond or increases the reagent that described peptide accumulates in cell, and wherein optionally described cell-penetrating peptides forms a part for the sequence of described peptide.
4. the peptide that uses of claim 3, wherein said cell-penetrating peptides is selected from the poly polyarginine-tag with sequence RRRRRRRRR (SEQ ID NO:46), derived from NGR peptide (CNGRCG:SEQ ID NO:47) or feeler foot leader peptide (CT) (KKWKMRRNQFWVKVQRG:SEQ ID NO:48) of aminopeptidase (CD13) N part, Bcl-2 binding peptide (caprinoyl-KNLWAAQRYGRELRRMSDEFEGSFKGL:SEQ ID NO:49), tat sequence (RKKRRQRRR:SEQ ID NO:50), Bufo siccus antibacterial peptide (TRSSRAGLQFPVGRVHRLLRK:SEQ ID NO:51), the fragments of peptides (TRRQRTRRARRNR:SEQ ID NO:52) of human T lymphotrophic virus (HTLV) II type Rex, adipose membrane translocating peptides (KKAAAVLLPVLLAAP:SEQ ID NO:53), the NRP-1 targeting peptides (RPARPAR:SEQ ID NO:54) of streptomyces hygroscopicus (Streptomyces hygroscopicus) and penetrate element (RQIKIWFQNRRMKWKKGG:SEQ ID NO:55).
5. the peptide used any one of aforementioned claim, described peptide has sequence tat-PPTVPTR (SEQ ID NO:39) or sequence (R) 9-PPTVPTR (SEQ ID NO:40).
6. a compositions, it comprises the peptide any one of claim 1 or 5 for the treatment of effective dose.
7. a pharmaceutical composition, it comprises compositions and one or more pharmaceutically acceptable carriers of claim 6.
8. the peptide any one of claim 1-5; or the compositions of claim 6 or 7; be used for the treatment of and/or prevent the method for blood vessel or cardiovascular disease, cancer, the disease relevant to UV radiation, respiratory system disease, neurodegenerative disease, inflammatory diseases or gastrointestinal disease, and/or wherein said peptide is used for defying age or protects reagent as light.
9. the peptide that uses of claim 8 or compositions, wherein said disease is selected from angiogenesis dependent disease, dermatosis, allergy and autoimmune disease, hematologic disease, muscle disease, joint disease, gastrointestinal disease and the disease relevant to impaired metabolism.
10. for peptide or the compositions of claim 8, wherein said cancer is skin carcinoma, cancerate before skin injury, squamous cell carcinoma, colon cancer, adenocarcinoma of colon, carcinoma of prostate, leukemia, SMP, neuroblastoma, ganglioneuroma, optimum or malignant papilloma.
Peptide any one of 11. claim 1-5, or the compositions of claim 6 or 7, be used for the treatment of and/or prevent skin injury that dermatosis, UV skin injury, the dermatosis caused by aging, ROS induce, cross presenility, UV (UV A or UV B) expose after the cutaneous tumor method of skin injury that occurs or caused by spawn (such as causing ROS increase or cause the product of inflammation).
Peptide any one of 12. claim 1-5, or the compositions of claim 6 or 7, wherein nadph oxidase 1 (Nox1) activity reduces at least 20% or at least 30% or at least 35%, 40%, 45%, 50%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, and/or wherein said peptide selectivity reduces and/or suppress nadph oxidase 1 (Nox1) active.
Peptide any one of 13. claim 1-5; or the compositions of claim 6 or 7; for resisting the guard method of UV radiation or for strengthening the method protected the light of UVA and UVB two kinds of rays, it comprises the skin described peptide of effective dose or described compositions being given or be administered to experimenter.
The peptide of 14. claim 13 or compositions, wherein said experimenter is easy to the risk being in the skin carcinoma caused because long-term UVA and UVB exposes.
Peptide any one of 15. claim 1-5, or the compositions of claim 6 or 7, be used for the treatment of and/or repair and/or nurse the method for the skin of experimenter, mucosa, scalp and/or hair.
Peptide any one of 16. claim 1-5, or the compositions of claim 6 or 7, for reducing, postponing and/or anti-aging, photoaging and the method because of the sign of crossing the dermatosis that presenility causes in advance, it comprises and described peptide or compositions to be given or to be administered to skin.
Peptide any one of 17. claim 1-5, or the compositions of claim 6 or 7, for (i) nursing, repair and protection skin, mucosa, scalp and/or hair to resist UV radiation, UV damage, old and feeble, or resist any such as increased by ROS or inflammation increase and induction stress, the reagent of inflammation, skin injury; Or (ii) reduces, postpones and/or anti-aging, photoaging and the method because of the sign of crossing the dermatosis that presenility causes in advance.
18. 1 kinds of generations for the level and/or inhibit activities oxygen clusters that reduce reactive oxygen species in experimenter in need, and/or the method that selectivity reduces and/or suppresses nadph oxidase 1 (Nox1) active, it comprises the compositions that the peptide that gives to limit any one of claim 1-5 or claim 6 or 7 limit.
The method of 19. claim 18, wherein nadph oxidase 1 (Nox1) activity is reduced at least 20% or at least 30% or at least 35%, 40%, 45%, 50%, 54%, 55%, 56%, 57%, 58%, 59% or 60% by selectivity.
20. 1 kinds for providing the protection of resisting UV radiation or strengthening the method that the light for UVA and UVB two kinds of rays protects, it comprises the skin that compositions that the peptide that limits any one of the claim 1-5 by effective dose or claim 6 or 7 limits gave or be administered to experimenter.
21. 1 kinds of treatments and/or repair and/or the method for the nursing skin of experimenter, mucosa, scalp and/or hairs, it compositions comprised the peptide limited any one of claim 1-5 or claim 6 or 7 limit gives or is administered to skin.
22. 1 kinds of minimizings, postpone and/or anti-aging, photoaging and the method because of the sign of crossing the dermatosis that presenility causes in advance, it compositions comprised the peptide limited any one of claim 1-5 or claim 6 or 7 limit gives or is administered to skin.
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